In the field of gene recombination or the field of medicine, for introduction of a gene into cells of mammals including human, physical methods using electroporation or metal microparticles, chemical methods using nucleic acid, polycation, or liposome, and biological methods using vectors for gene transfer wherein viruses are used as the vectors (hereinafter, referred to as viral vectors) have been currently used. The vectors for gene transfer wherein viruses are used as the vectors mean vectors obtained by altering natural viruses so that the viruses can transfer a desired gene or the like into a target, and the development of such vectors has been recently advanced. Vectors prepared by gene recombination technology are usually called recombinant viral vectors. Well-known examples of vectors which the recombinant viral vectors are derived from include viruses with envelopes, for example retrovirus, lentivirus, Sendai virus, and herpes virus, and viruses without envelopes, for example adenovirus, and adeno-associated virus (hereinafter, referred to as AAV).
In particular, AAV can infect a wide variety of cells including human cells, and AAV infects even non-dividing cells in which differentiation terminates, including blood cells, muscle cells, and nerve cells. In addition, since AAV is not pathogenic to human, it has a low risk of adverse effect. The viral particle of AAV is physicochemically stable. For these reasons, AAV has recently attracted attention to utility value as a vector for gene transfer used in gene therapy for the treatment of congenital genetic disease as well as the treatment of cancer or infection.
A method of producing a gene recombinant viral vector usually comprises introducing elements essential for formation of a viral particle in the form of a nucleic acid construct(s) into a cell to produce a cell having the ability to produce a virus (hereinafter, referred to as a virus-producing cell), and culturing the cell to express the elements essential for formation of the viral particle. In general, of the elements essential for formation of the viral particle, the elements that need to be provided in cis and the elements that can be provided in trans are separately introduced into a cell for viral production, thereby production of a wild-type virus and self-replication of a virus in a host infected with the virus are prevented (Patent Literature 1).
Hereinafter, as an example, a gene recombinant viral vector derived from AAV (hereinafter, referred to as rAAV) is specifically explained. An established method of producing the viral vector comprises 1) introduction of a rAAV plasmid in which an ITR placed at each end of the wild-type AAV genome is left and rep and cap genes are removed, 2) introduction of a plasmid for expression of rep and cap genes to provide Rep and Cap proteins in trans, and since AAV needs provision of supplemental elements from a so-called helper virus such as an adenovirus, a herpesvirus, or a vaccinia virus for formation of the infectious viral particle, 3) infection with an adenovirus (Patent Literature 2). In addition, since a vector solution obtained by the above-mentioned method is theoretically contaminated with adenovirus, in order to avoid the adenovirus contamination, a method of producing a vector comprising, instead of the above-mentioned step 3), step 3′) introduction of a helper plasmid expressing only elements essential for formation of an AAV viral particle among adenovirus-derived elements (Helper-free system) has been developed (Patent Literature 3).
Virus-producing cells that have accomplished viral production are collected and homogenized to obtain a cell homogenate containing rAAV vectors. The cell homogenate is subjected to a suitable step such as filtration with a filter, ultracentrifugation, chromatography, or ultrafiltration to purify the rAAV vectors as a final product.
At present, as use of viral vectors is extended to the field of basic research or clinical application for gene therapy, a method of obtaining a viral vector with higher titer and higher purity is needed. Various improved methods are disclosed. For example, a method of enhancing viral production and a release rate of the virus into a culture supernatant which comprises culturing a virus-producing cell under a stress condition in which a culture medium has an elevated pH is known (Patent Literature 1). Other methods comprise improvement of steps on and after purification of a produced virus. On the other hand, there is still room for improvement in treatment of a virus-producing cell performed before purification of the virus.